CN115181166A - Soluble expression method of O-type foot-and-mouth disease virus VP1 protein - Google Patents

Abstract

The invention discloses a soluble expression method of O-type foot-and-mouth disease virus VP1 protein, which takes KM243095.1 gene published by GenBank as a reference sequence, optimizes a target gene sequence according to escherichia coli codon tropism, and synthesizes a target gene; cloning a target gene into a pET32a (+) prokaryotic expression vector, transferring into escherichia coli to construct pET32a-VP1 recombinant plasmid, then performing inducible expression of pET32a-VP1 recombinant protein, and finally purifying by using a nickel column. According to the invention, partial optimization is carried out on the gene sequence of the O-type FMDV according to the codon tropism of escherichia coli, the advantages of a T7 promoter, a His purification tag and a Trx solubility promoting tag are fully utilized, and the soluble expression of recombinant protein is realized and the purification efficiency is improved by combining codon optimization, his tag and terminator introduction at a carboxyl terminal and optimization of induced expression conditions.

Description

Soluble expression method of O-type foot-and-mouth disease virus VP1 protein

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a soluble expression method of O-type foot-and-mouth disease virus VP1 protein.

Background

Foot-and-mouth disease is an acute, hot, highly contagious disease caused by infection of cloven-hoofed animals with Foot-and-mouth disease virus (FMDV). Cattle, pigs, goats, sheep and the like in livestock are all susceptible animals and seriously harm the health development of animal husbandry and public health safety, the cattle, the pigs, the goats, the sheep and the like are listed as animal epidemic diseases in China, and the main method for controlling the disease at present is the combination of antibody detection and vaccine immunity. FMDV belongs to the foot-and-mouth disease virus genus of the picornaviridae family, mainly has 7 serotypes, namely A, C, O, SATI, SAT2, SAT3 and AsiaI types, FMDV of different serotypes has no cross protection, serotypes causing domestic animal diseases mainly have three serotypes of O, A and AsiaI, and O type is most popular. The VP1 protein is the main structural protein of the type O FMDV, contains most of the neutralizing antigen sites which stimulate the organism to produce neutralizing antibodies, so that the VP1 antibody is the main neutralizing antibody produced by FMDV immunization, and is an important basis for evaluating the immune effect of the vaccine.

The prokaryotic expression system is the most widely applied method for expressing protein at present and is the most classical method for expressing protein, and has the advantages of high gene expression amount, fast growth and propagation, short culture period, low cost, large-scale production and the like, but the expression form of exogenous genes expressed by the Escherichia coli prokaryotic expression system is mostly inclusion body expression, and although the expression has the advantage of high expression amount, the defects are that the expressed protein is often folded incorrectly due to too fast protein expression, so that the activity of the protein is lost, the purified protein can be used only after renaturation, and the renaturation efficiency of the protein is not high.

Disclosure of Invention

The invention aims to provide a soluble expression method of VP1 protein of O-type foot-and-mouth disease virus, which aims to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: a soluble expression method of O-type foot-and-mouth disease virus VP1 protein comprises the following steps:

step1, FMDV-O-VP1 protein gene synthesis: according to the KM243095.1 gene published by GenBank as a reference sequence, the O CATHAY isolate is the main topological type of O-type foot-and-mouth disease virus which is popular in swinery in China, and the structural protein VPl of the virus is exposed on the surface of virus particles and is the main component for inducing and generating neutralizing antibodies, and the protein expressed by the target gene can be used for the subsequent research and development of antibodies for detection and treatment. As a set of common codons adapted to Escherichia coli is formed in the evolution process, amino acids are transferred to ribosomes by tRNA in the protein translation process, and when the expression amount of tRNA corresponding to codons of an exogenous gene in host cells is low, the expression efficiency of protein is influenced, a target gene KM243095 is synthesized by optimizing a target gene sequence according to the preference of Escherichia coli to the common codons, namely codon tropism, and a BamHI enzyme cutting site, an XhoI enzyme cutting site, a His tag and a terminator are introduced into the gene KM243095. The enzyme digestion site is used for connecting the target gene to the carrier, and the His tag and the terminator are introduced to the carboxyl terminal, so that the exposure of the tag is facilitated, and the effect is better when the protein is purified by a nickel column subsequently;

construction of Step2 and pET32a-VP1 recombinant plasmids: recombining a target gene into a pET32a (+) vector, wherein the pET32a (+) vector contains a T7 promoter, his and a Trx label, and can remarkably promote the expression quantity and solubility of expressed target protein, transforming a plasmid into escherichia coli BL21 (DE 3) competent cells after recombination, coating the competent cells on an LB flat plate containing ampicillin resistance, culturing for 12h at 37 ℃, selecting a single colony, inoculating the single colony into an LB culture medium containing ampicillin, culturing for 6h at 37 ℃, centrifuging and collecting thalli at 12000r/min, extracting the plasmid, carrying out enzyme digestion identification on the plasmids, and sequencing;

step3, FMDV-O-VP1 protein induced expression: transferring the cultured bacterial liquid to LB culture medium containing ampicillin resistance, culturing at 200r/min and 37 deg.C to OD ₆₀₀ Stopping culturing when the culture temperature reaches 0.6-0.8; adding IPTG into a part of bacterial liquid for induction expression, centrifuging the induced bacterial liquid at 8000r/min for 5min, washing the precipitate for 1 time by using ultrapure water, then resuspending the precipitate by using a PBS solution, boiling the precipitate at 100 ℃ for 10min, carrying out mass induction after optimal induction conditions are determined by gel running, collecting bacterial precipitate, carrying out ultrasonic crushing in an ice bath, determining a protein expression form by gel running, and expressing the supernatant;

step4, FMDV-O-VP1 protein purification: passing the supernatant through 0.45 μm filter membrane, balancing nickel column with balance solution, loading, washing target protein with imidazole of different concentrations, collecting elution peak, determining elution concentration of target protein by running gel, and ultrafiltering and concentrating.

Preferably, in Step1, the target gene coding sequence consists of 639bp bases, and the optimized sequence is as follows: TAC is optimized to TAT, AAG is optimized to AAA, GAG is optimized to GAA, GAC is optimized to GAT, and the nucleotide series number of the target gene formed after final optimization is specifically as follows: <xnotran> ACCACCTCTGCGGGCGAATCTGCGGATCCCGTGACCACCACCGTCGAAAATTATGGTGGTGAAACACAAGCCCAGAGGCGCCAGCACACGGATATAGCGTTCATACTGGATAGGTTCGTAAAAGTCAAACCAAAAGAACAAGTCAATGTATTGGATTTGATGCAGATCCCTGCCCACACCTTGGTAGGGGCGCTCCTGCGAACGGCCACCTATTATTTCTCTGATCTGGAACTGGCCGTCAAACACGAAGGCGATCTCACCTGGGTCCCAAACGGTGCCCCCGAAACAGCACTGGATAACACTACCAACCCAACAGCTTATCACAAAGAACCGCTCACAAGGTTGGCACTGCCTTATACGGCTCCCCACCGCGTCTTAGCGACCGTCTATAACGGGAGCAGTAAATATGGTGATGCCAGCGCTAACAACGTGAAAGGTGACCTTCAAGTGCTGGCTCAGAAGATAGAAAAAACTCTACCTACCTCCTTCAACTTCGGTGCCATTAAAGCAACCCGTGTGACTGAACTGCTCTATAGGATGAAAAGAGCCGAAACGTATTGTCCCAGGCCCCTTCTCGCCATTCAACCGAGTGATGCTAGACACAAACAAAAAATTGTGGCACCCGCAAAACAGCTTCTG. </xnotran>

Preferably, in Step3, the final concentration of IPTG is 1mmol/L, and the condition for inducing expression is that the expression is induced for 2h at 37 ℃.

Preferably, in Step3, the ice-bath ultrasonication is carried out under the conditions of 250W of power, 4s of ultrasonication and 4s of stopping ultrasonication, the ultrasonication is carried out for 30min, a bacterium solution is clarified and transparent, and the centrifugation is carried out for 10min at 12000r/min after the ultrasonication.

Preferably, in Step4, the imidazole elution concentration is 150mM.

Preferably, in Step4, the specific process for FMDV-O-VP1 protein purification is as follows: balancing, loading, balancing for the second time, washing impurities, eluting, and concentrating by ultrafiltration.

Compared with the prior art, the invention provides a soluble expression method of the VP1 protein of the O-type foot-and-mouth disease virus, which has the following beneficial effects:

(1) The invention fully utilizes the advantages of the T7 promoter, the His purification tag and the Trx dissolution promotion tag in the pET32a vector, the T7 promoter is controlled by T7 RNA polymerase, the speed of synthesizing mRNA by the high-activity T7 RNA polymerase is 5 times faster than that of the Escherichia coli RNA polymerase, the expression quantity of the target protein can be improved to a great extent after induction expression, and the effect of promoting expression is achieved. His is a highly soluble polypeptide, which greatly increases the solubility of the target protein when expressed in fusion with the target protein. Meanwhile, the target protein and the His tag are expressed together, so that the purification of the target protein is facilitated, and similarly, the Trx solubility-promoting tag can also obviously promote the solubility of the expressed target protein. Based on the characteristics, the invention does not additionally add other solubilizing labels, and compared with the prior invention, the invention optimizes the target gene sequence by codons, avoids influencing the expression efficiency of the protein when the expression quantity of tRNA corresponding to the codon of the exogenous gene in a host cell is low, thereby improving the expression speed and the expression quantity of the target gene in escherichia coli. The optimization of the induction expression conditions is combined, wherein the optimization of the codons can improve the expression speed and the expression quantity of the target gene in the escherichia coli, and the optimization of the induction expression conditions can obtain more soluble expression proteins, so that the soluble expression of the recombinant proteins is realized, and the purification efficiency is improved.

(2) The invention also adds His label at the carboxyl terminal of the target protein and introduces terminator, which is more favorable for the exposure of the label compared with the introduction at the amino terminal, so that the effect is better when the protein is purified by nickel column subsequently.

Drawings

FIG. 1 is a drawing showing the result of identifying a recombinant plasmid PET32a-VP1 provided by Gene Synthesis Inc. in the example of the present invention, in which M: a 1kb ladder (10000, 8000, 6000, 5000, 4000, 3000, 2000, 1500, 1000, 500); 1: before enzyme digestion; 2: after digestion with BamHI and XhoI.

FIG. 2 is a diagram showing the results of the induction expression of the PET32a-VP1 recombinant plasmid provided by the embodiment of the present invention in E.coli, wherein M: protein molecular mass standard; 1: before IPTG induction; 2-4: 1mM IPTG 2, 3, 4 hours after induction.

FIG. 3 is a graph showing the results of identifying the expression pattern induced by FMDV-O-VP1 recombinant protein, wherein M: protein molecular mass standard; 1: crushing and then clearing the supernatant; 2: and precipitating after crushing.

FIG. 4 is a diagram showing the result of SDS-PAGE of column purification of FMDV-O-VP1 recombinant protein provided in the example of the present invention, in which M: protein molecular mass standard; 1: before the column; 2: flowing through the liquid; 3-5: 50. 150, 300mM imidazole eluent.

FIG. 5 is a graph showing the result of SDS-PAGE of the ultrafiltration concentration of FMDV-O-VP1 recombinant protein according to the present invention, in which M: protein molecular mass standard; 1: after ultrafiltration and concentration, FMDV-O-VP1 recombinant protein is obtained.

Detailed Description

The invention is described in further detail below by means of specific embodiments with reference to the attached drawings. It is to be understood that the embodiments described herein are illustrative only and are not limiting upon the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.

1. Materials and methods

1. Material

PET32a-VP1 gene synthesis services were purchased from Jinwei, suzhou, protein Marker from Baiolanbo, isopropyl-. Beta. -D-thiogalactopyranose (IPTG), ampicillin from Alantin, yeast extract, peptone from OXOID, sodium chloride, agar powder, potassium chloride, SDS, triton X-100, glycerol, KCL, KH2PO4, EDTA.2Na, naH2P 04.2H20, na2 HPO4.12H20 from Gum, ultrafiltration tubes from Millipore, SDS-precast gels from Kingosiri, tris-base, 1M Tris-HCl, pH8.0, 5 xloading buffer from white shark, MOPS, DTT from Laobao, bags from Rebbis, saudism filter from Sartuum, 5 lron nickel column from Redou.

2. Synthesis of FMDV-O-VP1 Gene

According to the KM243095.1 gene published by GenBank as a reference sequence consisting of 639bp bases, TAC is optimized to TAT, AAG is optimized to AAA, GAG is optimized to GAA, GAC is optimized to GAT according to the codon tropism of Escherichia coli, and BamHI enzyme cutting site, xhoI enzyme cutting site, his tag and terminator are introduced into the sequence for synthesizing VP1 gene by Jinzhi organism company of Suzhou.

3. Construction of FMDV-O-VP1 recombinant protein vector

Recombining a target gene into a pET32a (+) vector, transforming the target gene into escherichia coli BL21 (DE 3) competent cells, coating the escherichia coli BL21 (DE 3) competent cells on an LB plate containing ampicillin resistance, culturing the escherichia coli at 37 ℃ for 12 hours, selecting a single colony, inoculating the single colony into an LB culture medium containing ampicillin, culturing the bacterial colony at 37 ℃ for 6 hours, centrifuging the bacterial colony at 12000r/min, extracting a plasmid, carrying out enzyme digestion identification and sequencing on the plasmid, wherein the service is purchased from Jinzhi biology company, suzhou.

4. The FMDV-O-VP1 recombinant protein induces and expresses:

transferring the cultured bacterial liquid to 1L LB culture medium containing ampicillin resistance, stopping culturing when OD600 reaches 0.6-0.8 at 200r/min and 37 ℃, taking part of the bacterial liquid, adding IPTG (isopropyl-beta-D-thiogalactoside) to make the final concentration 1mM, carrying out induction expression, respectively sampling after 2h, 3h and 4h of induction, centrifuging the induced bacterial liquid at 8000r/min for 5min, washing the precipitate with ultrapure water for 1 time, then carrying out heavy suspension with PBS (phosphate buffered saline) solution, boiling at 100 ℃ for 10min, carrying out mass induction after determining the optimal induction condition by running SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), collecting bacterial precipitate, blowing and dissolving the precipitate with 40ml of bacterial lysate, carrying out ice bath ultrasonic crushing, wherein the ultrasonic condition is as follows: the power is 250W, the time is over 4s, the time is stopped for 4s, the crushing is carried out for 30min, and the bacterial liquid is clear and transparent. After being broken, the mixture is collected into a 50ml centrifuge tube and centrifuged at 12000rpm for 10min. Taking supernatant and precipitate, running SDS PAGE gel to determine protein expression form, wherein 40ul of crushed supernatant is taken, 40ul of crushed precipitate is re-suspended by 100ul 1xPBS, 10ul of each sample buffer (5 x) is added into the crushed precipitate, boiling is carried out for 10min, after instantaneous centrifugation, 12% SDS PAGE gel is used, and 140V constant pressure electrophoresis is carried out.

5. FMDV-O-VP1 protein purification

1) Balancing

The medium was equilibrated with equilibration solution at 2.0ml/min until the baseline plateaus for 3-5 column volumes and then zeroed.

2) Sample loading

After the sample is filtered by a 0.45um filter membrane, the sample is loaded at the speed of 1ml/min, and the flow-through liquid is collected when the baseline starts to peak in the loading process.

3) Balancing

After the sample loading is finished, the balance solution is used for balancing 2.0ml/min until the baseline is stable, and then impurities are washed.

4) Washing sundries

The eluate was eluted with a gradient eluent (50 mM) at 2.0ml/min and the eluate was collected.

5) Elution is carried out

The eluates eluted with the 100mM,150mM,300mM 3 concentration gradients were eluted at 2.0ml/min, and the eluates were collected and run on SDS PAGE gel to determine the elution concentration of the target protein.

6) Concentrating by ultrafiltration

The solution was exchanged with 10kd ultrafiltration tube and 1 xPBS: 5ml of the harvest was centrifuged with 10ml of 1xpbs: centrifuging at 8000r/min for 10min, adding 10ml 1xPBS once for each separation, gently re-suspending, centrifuging for three times, completely changing the solution, detecting the concentration by BCA, and running SDS PAGE gel.

6. Establishment of method for detecting anti-O type foot-and-mouth disease virus VP1 protein antibody by indirect ELISA

1) Antigen coating

The FMDV-O-VP1 recombinant protein is diluted to 0.1 mu g-6.4 mu g/mL by CBS coating buffer solution, diluted by 4 times of gradient and mixed evenly. And adding the diluted solution into a microplate according to 100 muL/hole, covering a sealing membrane, incubating at 37 ℃ for two hours, taking out, washing 5 times by using 1 XPBST (pH7.4), 200 muL/hole.

2) Sealing plate

Adding RAC blocking liquid into the micropore plate according to 200 mu L/hole, incubating at 37 ℃ for 2h, taking out and directly patting to dry.

3) Serum incubation

The positive and negative serum samples were diluted with 1xPBS at 50-fold, 100-fold, 200-fold, 400-fold. Add 100ul,1xPBS to each well as blank, incubate at 37 ℃ for 30min, take out and wash plate 5 times.

4) HRP enzyme-labeled Secondary antibody incubation

The enzyme-labeled secondary antibody was diluted 3000 times with 1xPBS, added to each well at 100ul, and incubated at 37 ℃ for 30min. The plate was washed 5 times.

5) Adding substrate color developing liquid

Adding substrate developing solution into each well at a rate of 100 ul/well, and placing in a constant temperature incubator at 37 ℃ for 15min.

6) And (3) determination: add stop solution 50 μ L into each well, mix well by gentle shaking, measure the absorbance value at 450nm within 5 minutes.

7. And (4) testing the O-type foot-and-mouth disease negative and positive sample by using the optimized indirect ELISA method, wherein the steps are as above.

2. Analysis of results

1. Identification result of PET32a-VP1 recombinant plasmid

The constructed recombinant plasmid PET32a-VP1 is subjected to enzyme digestion identification by BamHI and XhoI, and a product is subjected to agarose gel electrophoresis, as shown in figure 1, the result shows that the linear plasmid before enzyme digestion is about 6500bp, and the target fragment after enzyme digestion is about 639bp, and the size is consistent with the expected size.

2. The result of exploring the induction expression condition of FMDV-O-VP1 recombinant protein

After 1mM IPTG induction at 37 ℃ for 2, 3 and 4 hours, SDS-PAGE results show that the molecular weight of the recombinant protein after induction is about 42kd, which is consistent with the size of the expected expression protein, and the optimal expression can be reached after 2 hours of induction, as shown in figure 2.

3. Determination of FMDV-O-VP1 recombinant protein induced expression form

The supernatant and the precipitate obtained by ultrasonication after the induction expression of Escherichia coli were run on SDS-PAGE, and the results are shown in FIG. 3, in which the target protein was expressed in the supernatant.

4. FMDV-O-VP1 recombinant protein column purification

The FMDV-O-VP1 recombinant protein is mainly present in 150mM imidazole eluate.

5. FMDV-O-VP1 recombinant protein ultrafiltration concentration

The concentration of FMDV-O-VP1 recombinant protein purified by the method disclosed by the invention is 5.34mg/mL, about 5mL is obtained, and the SDS-PAGE running gel shows that the protein purity is better.

6. And (3) an optimized result of a method for detecting the VP1 protein antibody of the O-type foot-and-mouth disease virus by indirect ELISA.

TABLE 1 ELISA detection OD of different antigen coating concentration and different serum dilution times _450nm Value of

Note: blank OD was 0.058.

The maximum dilution of VP1 antigen and positive control serum, in which the OD450nm value of the positive serum wells is close to 1.0 and P/N is not less than 2.0 (the OD450nm value of the serum sample wells is equal to the blank well OD450nm value)/(the OD450nm value of the negative control wells is equal to the blank well OD450nm value), is used as the optimum working concentration of VP1 antigen and positive serum. As can be seen from the above table, the optimized plate concentration was determined to be 0.4. Mu.g/mL, the sample dilution concentration was determined to be 100-fold dilution, and the plate was determined to be positive with OD > 0.3.

7. Result of detecting actual sample by indirect ELISA anti-O type foot-and-mouth disease virus VP1 protein antibody

30 parts of pig serum with negative and positive VP1 antibodies of the known foot-and-mouth disease are detected by an optimized indirect ELISA method, and the coincidence rate reaches 96.7 percent.

3. Conclusion

According to the invention, partial optimization is carried out on the gene sequence of the O-type FMDV according to the tropism of escherichia coli, the advantages of a His purification tag and a Trx dissolution promotion tag are combined, so that FMDV-O-VP1 recombinant protein is subjected to soluble expression, the prokaryotic expression efficiency is greatly improved, the recombinant protein is subsequently used for plate wrapping, an ELISA detection method for the O-type foot-and-mouth disease virus VP1 protein antibody is established, and the positive coincidence rate of the detected actual sample is up to 96.7%.

The above description is intended to be illustrative of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Claims (6)

1. A soluble expression method of O-type foot-and-mouth disease virus VP1 protein is characterized by comprising the following steps:

step1, FMDV-O-VP1 protein gene synthesis: according to a reference sequence of KM243095.1 published by GenBank, optimizing a target gene sequence according to codon tropism of escherichia coli, synthesizing a target gene, and introducing a BamHI enzyme cutting site, an XhoI enzyme cutting site, a His tag and a terminator into the target gene;

construction of Step2 and pET32a-VP1 recombinant plasmids: recombining a target gene to a pET32a (+) vector, transforming the target gene to escherichia coli BL21 (DE 3) competent cells, coating the escherichia coli BL21 (DE 3) competent cells on an LB (Lubtinum-benzyl-penicillin) -resistant plate, culturing for 12h at 37 ℃, selecting a single colony, inoculating the single colony into an LB culture medium containing ampicillin, performing centrifugation at 200r/min, culturing for 6h at 37 ℃, performing centrifugation at 12000r/min to collect thalli, extracting plasmids, performing enzyme digestion identification on the plasmids, and sequencing;

induced expression of Step3 and FMDV-O-VP1 proteins: transferring the cultured bacterial liquid to LB culture medium containing ampicillin resistance, culturing at 200r/min and 37 deg.C to OD ₆₀₀ Stopping culturing when the culture temperature reaches 0.6-0.8; adding IPTG into a part of bacterial liquid for induction expression, then centrifuging the induced bacterial liquid at 8000r/min for 5min, washing the precipitate for 1 time by ultrapure water, then resuspending by a PBS solution, boiling at 100 ℃ for 10min, carrying out gel running to determine the optimal induction conditions, then carrying out mass induction, collecting bacterial precipitate, carrying out ice bath ultrasonic crushing, determining the protein expression form by gel running, and carrying out supernatant expression;

step4, FMDV-O-VP1 protein purification: passing the supernatant through 0.45 μm filter membrane, balancing nickel column with balance solution, loading, washing target protein with imidazole of different concentrations, collecting elution peak, determining elution concentration of target protein by running gel, and ultrafiltering and concentrating.

2. The soluble expression method of VP1 protein of foot-and-mouth disease virus type O according to claim 1, wherein in Step1, the target gene coding sequence consists of 639bp bases, and the optimized sequence is: TAC is optimized to TAT, AAG is optimized to AAA, GAG is optimized to GAA, and GAC is optimized to GAT.

3. The soluble expression method of VP1 protein of foot-and-mouth disease virus type O according to claim 2, wherein IPTG final concentration is 1mmol/L in Step3, and the induction expression condition is 37 ℃ for 2h.

4. The soluble expression method of VP1 protein of foot-and-mouth disease virus type O according to claim 3, wherein in Step3, the ice bath ultrasonication conditions are 250W power, 4s ultrasonication, 4s rest, 30min fragmentation, clear and transparent bacterial solution, and centrifugation is performed at 12000r/min for 10min after fragmentation.

5. The method for soluble expression of VP1 protein of foot-and-mouth disease virus type O according to claim 1, wherein the elution concentration of imidazole in Step4 is 150mM.

6. The soluble expression method of VP1 protein of foot-and-mouth disease virus type O according to claim 5, wherein the specific process for FMDV-O-VP1 protein purification in Step4 is as follows: balancing, loading sample, balancing for the second time, washing impurity, eluting, and concentrating by ultrafiltration.

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